The present invention relates to a device for the detection of an analyte in a sample and to a method of detecting the presence of an analyte in a sample.
Current technologies used in the diagnostic industry require large expensive equipment for the detection of analytes. For example, immunoassays require gamma-detectors, spectrophotometers, lasers, etc. and DNA detection after PCR processes requires electrophoresis and absorption methods, all of which depend on the specific probe used for signal amplification.
A number of devices have been described in the literature which have been designed for simple single-step assays and make use of area separation to carry out the different reactions and washing steps required. For example, antibody-based tests such as the pregnancy testing device xe2x80x9cClearblue One-Stepxe2x80x9d by Unipath employ a wick to absorb urine which then travels the length of a pen-like device. The hormone hCG is captured by the first layer which contains mobile blue latex particles to which mAb has been coupled. The urine flow carries the latex, and bound hCG, to a second area containing immobilized mAb recognizing a second epitope site on the hormone. Any hCG bound to the latex will be prevented from continuing past the second area as evidence by a discrete blue line. In the absence of hCG, the latex moves through to a third area and captured by immobilized anti-Fc antibody. Other disposable devices use liquid-operated switch (illustrated in FIG. 12.7 Chapter 12 by A. P. H. Farnsworth, in xe2x80x9cMolecular and Antibody Probes in Diagnosisxe2x80x9d edited by M. R. Walker and R. Rapley, John Wiley and sons, 1993) to carry out sequential steps in the ELISA-type processes. In DNA-based technologies, a product for performing the multiple steps required in PCR technology has been released which by compartmentalizing the different steps in a single disposable device offers simplicity and reduction of cross-contamination of the PCR products.
In International Patent Application Nos. PCT/AU88/00273, PCT/AU89/00352, PCT/AU90/00025, PCT/AU92/00132, PCT/AU93/00590, PCT/AU93/00620 and PCT/AU94/00202 there is disclosure of biosensors which can be used to detect analytes. The disclosure of these documents is included herein by cross-reference.
It is believed that by adapting these biosensors and existing diagnostic techniques improved detection devices and methods of detection can be achieved.
Accordingly in a first aspect the present invention consists in an analyte detection device comprising first and second zones, means to allow addition of a probe to the first zone, means to allow addition of a sample suspected to contain an analyte to the first zone, and means to allow passage of the probe from the first zone to the second zone; the first zone containing ligands reactive with the analyte and the second zone including a membrane the impedance of which is dependent on the presence or absence of the probe and means to measure the impedance of the membrane.
The means to allow addition of the probe and sample to the first zone may be the same or different.
In a preferred embodiment of the present invention the probe includes an ionophore, preferably gramicidin.
In a further preferred embodiment of the present invention the membrane comprises a first and second layer of closely packed arrays of amphiphilic molecules and a plurality of ionophores comprising a first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer, the second half membrane spanning monomers being capable of lateral diffusion within the second layer independent of the first half membrane spanning monomers, the first half membrane spanning monomers being prevented from lateral diffusion in the first layer, and a second ligand provided on at least the second half membrane spanning monomers, said second ligand being reactive with the probe or a portion thereof, the binding of the probe to the second ligand causing a change in the relationship between the first half membrane spanning monomers and the second half membrane spanning monomers such that the flow of ions across the membrane via the ionophores is allowed or prevented, and measuring the impedance of the membrane.
In yet another preferred embodiment the ligands in the first zone are antibodies or binding fragments thereof.
In a second aspect the present invention consists in a method of detecting the presence of an analyte in a samnple, the method comprising contacting the sample with a carrier including a plurality of first ligands reactive with the analyte to allow binding of the analyte to the carrier ligands, contacting the carrier with a membrane comprising a first and second layer of a closely packed array of amphiphilic molecules and a plurality of ionophores comprising a first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer, the second half membrane spanning monomers being capable of lateral diffusion within the second layer independent of the first half membrane spanning monomers, the first half membrane spanning monomers being prevented from lateral diffusion in the first layer, and a second ligand provided on at least the second half membrane spanning monomers, said second ligand being reactive with the analyte or a portion thereof, the binding of the analyte to the second ligand causing a change in the relationship between the first half membrane spanning monomers and the second half membrane spanning monomers such that the flow of ions across the membrane via the ionophores is allowed or prevented, and measuring the impedance of the membrane.
The first half membrane spanning monomer in the first layer may be prevented from diffusing laterally using any of a number of known techniques, however, it is presently preferred that the monomer and the amphiphilic molecules each include or are decorated with at least one moiety cross-linked with at least one corresponding moiety on another of these molecules. Under appropriate stimulus, such as W radiation or ionizing radiation, the cross-linkable moieties can be caused to polymerize thereby resulting in the membrane being cross-linked in one layer.
The first half membrane spanning monomers may also be prevented from diffusing laterally by selecting lipids for the first layer of the membrane which are crystalline at room temperature. This eliminates lateral diffusion in the first layer.
In a further preferred embodiment of the present invention the first half membrane spanning monomers in the first layer are prevented from diffusing laterally by fixing the first layer and the monomers therein to a solid support. This may be achieved by providing groups on the amphiphilic molecules in the first layer and on the monomers therein which are reactive with the solid support or with corresponding groups provided thereon.
In another prefered form of the invention a proportion of the amphiphlic molecules are membrane spanning amphiphiles, the membrane spanning amphiphiles being archeobacterial lipids or tail to tail chemically linked bilayer amphiphiles. It is also preferred that the half membrane spanning monomers are gramicidin monomers.
In yet another preferred embodiment the membrane includes a plurality of third ligands attached to amphiphiles in the membrane, preferably membrane spanning amphiphiles. These third ligands are preferably prevented from diffusing laterally within the membrane. In the device of the first aspect of the present invention these third ligands will be reactive with probe or a portion thereof, whilst in the method of the second aspect of the present invention they will be reactive with the analyte.
The ligands may be the same or different and are preferably selected from the group consisting of polyclonal or monoclonal antibodies, antibody fragments including at least one Fab fragment, antigens, lectins, haptens, chelating agents and dyes.
The ligands are preferably attached to the ionophores and/or membranes via linkers. Suitable linkers are set out in PCT/AU90/00025, PCT/AU92/00132 and PCT/AU93/00509.
As will be recognized by those skilled in this field it is preferable that the membrane is attached to an electrode such that a reservoir exists between the electrode and the membrane. Molecules and methods by which this may be readily achieved are set out in PCT/AU92/00132 and PCT/AU93/00509. As stated above the disclosures of these documents are incorporated by cross reference.
In a third aspect the present invention consists in an analyte detection device comprising:
membrane including ligands reactive with an analyte;
means to measure the impedance of the membrane; and
means to move an analyte bound to the ligands away from the membrane without disrupting the binding of the ligands to the analyte;
wherein the movement of the analyte away from the membrane causes a change in the impedance of the membrane.
In a preferred embodiment of this aspect of the present invention the analyte is bound to a carrier via a plurality of second ligands. Preferably the carrier is a bead, or a charged or magnetic particle.
In a preferred embodiment the means to move the analyte comprises an electric field, magnetic field or liquid flow.
In another preferred embodiment the membrane ligands are attached to amphiphiles of the membrane, movement of the analyte causing extraction of the ligands and attached amphiphiles from the membrane.
In yet another preferred embodiment the membrane ligands are attached to ionophores within the membrane, movement of the analyte causing extraction of the ligands and attached ionophores from the membrane. The ionophores are preferably gramicidin.
In a fourth aspect the present invention consists in a method of determining the presence or absence of an analyte in a sample, the method comprising adding the sample to the device of the first or third aspect of the present invention and measuring a changing conductivity or capacitance of the membrane.
In a preferred embodiment of the present invention the membrane is as described in International Patent Application Nos. PCT/AU88/00273, PCT/AU89/00352, PCT/AU90/00025, PCT/AU92/00132, PCT/AU93/00590, PCT/AU93/00620 or PCT/AU94/00202.